I'm Nate Wideman. I like reading and I do Cross Country in the fall. I will be entering 9th Grade next year. I am interested in biochemistry and the uses of DNA as information storage and as small scale building material. Sometimes I like to randomly take on a subject and see if I could handle it. Most of the time I end up with headaches and no progress, but chemistry stuck well enough for me to (partially) understand and enjoy it. I found myself leaning towards organic chem because the molecules tended to be larger and more interesting.
Nathaniel Wideman 1/7/2017 Vaccine Producing Bacteria Design Purpose: Yellow fever outbreaks are present in forty seven countries, in South America and Africa. It is a virus spread by mosquitoes and can cause fever, muscle pain, headache, loss of appetite, nausea, or vomiting, though many do not experience symptoms. The World Health Organization estimates it was responsible for 29,000-60,000 deaths in 2013. The WHO has worked with the governments of fourteen countries to eliminate yellow fever outbreaks. The vaccine can be used at a fractional dose (a fifth) of the amount at which it is currently being administered and still prevent infection for life without any further vaccinations. The WHO estimates that 80% of the population needs to be vaccinated in order to prevent disease outbreak. The United States, which has not seen outbreaks on its soil, vaccinates citizens going to these countries to prevent the disease from coming back with them. However; costs and obstacles of vaccine production have caused a shortage in vaccines, and the supply is predicted to run out by midsummer, 2017. The vaccines need to made in a cheaper manner.
Competing Technologies: Without genetic engineering, vaccines are usually made by taking sterilized chick embryo cells and infecting them. The virus infects the chick cells, grows, and multiplies without other viruses or bacteria stopping it. Once they have reproduced enough, they are separated from the chick cells and harvested. Some vaccines are just the antigen, or the surface protein of the bacteria. This is like a mug shot of a criminal, it tells the immune system what the virus looks like yet poses no threat. Other vaccines have the entire outer shell, but none of the RNA or DNA that allows the virus to reproduce. Genetic Engineering has allowed bacteria, plants, even hamster cells to produce vaccines. They are just proteins, whose genetic code is spread between victims and easily accessible. Zika, Ebola, and Hepatitis B vaccines can all be produced by GMOs. This makes them cheaper and easier to make (bacteria free chick embryo cells are expensive). The simplest versions are the ones that only make the surface protein. It is a single protein that can be easily ejected. But the many proteins that make the outer shell, which are designed to self assemble inside the cell, can be harder to eject. Yellow fever vaccines are as such.
The Design:
The idea behind this is to have a bacteria, which will be a chassis with minimal DNA, make the vaccine inside of itself, then rupture its cell wall, sidestepping the problem of getting the vaccine out. The bacteria still multiply, but once they reach a certain number, they produce and die instead. Their numbers plummet and they begin to multiply again, keeping their population sustained while still making the vaccine. This requires Quorum Sensing, the bacteria need to be able to know how dense the population is, and how much of the vaccine is being made. The former is present in all bacteria, and the latter can be made by inserting the Quorum Sensing of another species of bacteria. The modified bacteria would have two Quorum Sensors on its surface (not counting the one universal to all bacteria). The first would be its native quorum sensor, the second one added from Clostridium tetani. The reason this bacteria, one responsible for tetanus, is being used is because it has been nearly wiped out, preventing cross talk between species. Its communication line has been left open. The signal protein for this line, in the modified bacteria, would not be made constantly by the bacteria. Instead the gene would be triggered by the same promoters that make the vaccine. By using this signal, the bacteria can communicate when they have made a vaccine and know how many are being made.
Vaccine Production
Signal Protein Production
0
0
1
1
As mentioned earlier, the bacteria has two “modes,” make vaccines or divide. This is controlled by the inputs from the Quorum sensors. Bacteria divide unless the number of bacteria is high and the number of vaccines is low. The logic gate is used is an ORN gate.
Bacterial Presence (Quorum sensing)
Signal Protein Presence (modified Quorum sensing)
Reproduction
0
0
1
1
0
0
0
1
1
1
1
1
Vaccine production is turned on only when Reproduction is turned off, so it is controlled with an ANDN gate.
Bacterial Presence (Quorum sensing)
Signal Protein Presence (modified Quorum sensing)
Vaccine Production
0
0
0
1
0
1
0
1
0
1
1
0
Once the vaccine builds up in the bacteria, the bacteria needs to get them into the open. Monitoring vaccine production, a third signal molecule follows the same truth table as the second. This one, however, stays within the bacteria. If in high enough concentrations, it activates an enzyme used by bacteriophages to leave an infected bacteria. This enzyme inhibits cell wall proteins and DNA, causing them to shut down. The cell wall falls apart, and the viruses, in nature, spread out to other bacteria. In the modified bacteria, the vaccines would simply spread out into the mixture.
Vaccine Bacterial Explosion.jpg
The vaccine can now extracted from the solution. The remaining bacteria are left to divide and make more vaccines, keeping the flow constant.
The Results: In the ideal environment, the bacteria would produce vaccines when in high concentrations, and double in low concentrations. This would mean the bacteria multiply if there were few of them and would produce vaccines if there were many. They would not, however, produce vaccines if vaccine concentrations and bacterial numbers were high. This would allow Pharmacists to harvest vaccines and still have a factory that could be grown and expanded.
Vaccine numbers
Bacterial numbers
Reproduction
Vaccine production
Low
Low
On
Off
Low
High
Off
On
High
Low
On
Off
High
High
On
Off
Potential Problems and Solutions:
There are a few things that could go wrong with the bacteria’s vaccine production and division. It is possible that all the bacteria could switch into making vaccines, all die, and leave no or very few bacteria left. The bacteria would be stripped of its amino-acid making proteins, and would have to receive them from the outside. It could not survive in the wild, but also might not survive in a lab, presenting another problem. It is possible that the bacteria would communicate with Clostridium tetani and trigger an early response. If the bacteria malfunctioned, it could be killed with the bacteriophage enzyme or starved of amino-acids. If it did not produce vaccines or did not rupture the DNA could be changed to make it more or less sensitive to signal proteins.
Testing:
As mentioned above, GFP could be attached to one of the bacteria’s vital proteins to see if it survived. An engineer could also forgo GFP and simply test to see if it survived in the environment it would be needed in. GFP could also be attached to the external signal proteins to see how much of the vaccine was being produced. Some testing would be needed to deduce how the bacteria would interact with tetanus, and what would happen if tetanus contaminated the solution Additional testing would be needed to insure that the bacteria don’t all commit suicide or don’t all stay alive, either of which would render it near useless. The former would hinder its continuous production, while the latter would prevent vaccines from being harvested out of the bacteria.
Nathaniel Wideman
1/7/2017
Vaccine Producing Bacteria Design
Purpose:
Yellow fever outbreaks are present in forty seven countries, in South America and Africa. It is a virus spread by mosquitoes and can cause fever, muscle pain, headache, loss of appetite, nausea, or vomiting, though many do not experience symptoms. The World Health Organization estimates it was responsible for 29,000-60,000 deaths in 2013. The WHO has worked with the governments of fourteen countries to eliminate yellow fever outbreaks. The vaccine can be used at a fractional dose (a fifth) of the amount at which it is currently being administered and still prevent infection for life without any further vaccinations. The WHO estimates that 80% of the population needs to be vaccinated in order to prevent disease outbreak. The United States, which has not seen outbreaks on its soil, vaccinates citizens going to these countries to prevent the disease from coming back with them. However; costs and obstacles of vaccine production have caused a shortage in vaccines, and the supply is predicted to run out by midsummer, 2017. The vaccines need to made in a cheaper manner.
Competing Technologies:
Without genetic engineering, vaccines are usually made by taking sterilized chick embryo cells and infecting them. The virus infects the chick cells, grows, and multiplies without other viruses or bacteria stopping it. Once they have reproduced enough, they are separated from the chick cells and harvested. Some vaccines are just the antigen, or the surface protein of the bacteria. This is like a mug shot of a criminal, it tells the immune system what the virus looks like yet poses no threat. Other vaccines have the entire outer shell, but none of the RNA or DNA that allows the virus to reproduce.
Genetic Engineering has allowed bacteria, plants, even hamster cells to produce vaccines. They are just proteins, whose genetic code is spread between victims and easily accessible. Zika, Ebola, and Hepatitis B vaccines can all be produced by GMOs. This makes them cheaper and easier to make (bacteria free chick embryo cells are expensive). The simplest versions are the ones that only make the surface protein. It is a single protein that can be easily ejected. But the many proteins that make the outer shell, which are designed to self assemble inside the cell, can be harder to eject. Yellow fever vaccines are as such.
The Design:
The idea behind this is to have a bacteria, which will be a chassis with minimal DNA, make the vaccine inside of itself, then rupture its cell wall, sidestepping the problem of getting the vaccine out. The bacteria still multiply, but once they reach a certain number, they produce and die instead. Their numbers plummet and they begin to multiply again, keeping their population sustained while still making the vaccine.
This requires Quorum Sensing, the bacteria need to be able to know how dense the population is, and how much of the vaccine is being made. The former is present in all bacteria, and the latter can be made by inserting the Quorum Sensing of another species of bacteria. The modified bacteria would have two Quorum Sensors on its surface (not counting the one universal to all bacteria). The first would be its native quorum sensor, the second one added from Clostridium tetani. The reason this bacteria, one responsible for tetanus, is being used is because it has been nearly wiped out, preventing cross talk between species. Its communication line has been left open. The signal protein for this line, in the modified bacteria, would not be made constantly by the bacteria. Instead the gene would be triggered by the same promoters that make the vaccine. By using this signal, the bacteria can communicate when they have made a vaccine and know how many are being made.
As mentioned earlier, the bacteria has two “modes,” make vaccines or divide. This is controlled by the inputs from the Quorum sensors. Bacteria divide unless the number of bacteria is high and the number of vaccines is low. The logic gate is used is an ORN gate.
(Quorum sensing)
(modified Quorum sensing)
Vaccine production is turned on only when Reproduction is turned off, so it is controlled with an ANDN gate.
(Quorum sensing)
(modified Quorum sensing)
Once the vaccine builds up in the bacteria, the bacteria needs to get them into the open. Monitoring vaccine production, a third signal molecule follows the same truth table as the second. This one, however, stays within the bacteria. If in high enough concentrations, it activates an enzyme used by bacteriophages to leave an infected bacteria. This enzyme inhibits cell wall proteins and DNA, causing them to shut down. The cell wall falls apart, and the viruses, in nature, spread out to other bacteria. In the modified bacteria, the vaccines would simply spread out into the mixture.
The vaccine can now extracted from the solution. The remaining bacteria are left to divide and make more vaccines, keeping the flow constant.
The Results:
In the ideal environment, the bacteria would produce vaccines when in high concentrations, and double in low concentrations. This would mean the bacteria multiply if there were few of them and would produce vaccines if there were many. They would not, however, produce vaccines if vaccine concentrations and bacterial numbers were high. This would allow Pharmacists to harvest vaccines and still have a factory that could be grown and expanded.
Potential Problems and Solutions:
There are a few things that could go wrong with the bacteria’s vaccine production and division. It is possible that all the bacteria could switch into making vaccines, all die, and leave no or very few bacteria left. The bacteria would be stripped of its amino-acid making proteins, and would have to receive them from the outside. It could not survive in the wild, but also might not survive in a lab, presenting another problem. It is possible that the bacteria would communicate with Clostridium tetani and trigger an early response. If the bacteria malfunctioned, it could be killed with the bacteriophage enzyme or starved of amino-acids. If it did not produce vaccines or did not rupture the DNA could be changed to make it more or less sensitive to signal proteins.
Testing:
As mentioned above, GFP could be attached to one of the bacteria’s vital proteins to see if it survived. An engineer could also forgo GFP and simply test to see if it survived in the environment it would be needed in. GFP could also be attached to the external signal proteins to see how much of the vaccine was being produced. Some testing would be needed to deduce how the bacteria would interact with tetanus, and what would happen if tetanus contaminated the solution Additional testing would be needed to insure that the bacteria don’t all commit suicide or don’t all stay alive, either of which would render it near useless. The former would hinder its continuous production, while the latter would prevent vaccines from being harvested out of the bacteria.
Inserted DNA sequence:
Design Power Point